Collagens are the major structural proteins in the extracellular matrix of animals and are defined by a characteristic triple-helix structure that requires a (Gly-Xaa-Yaa)n repeating sequence. The amino acids found in the Xaa and Yaa positions are frequently proline, where Pro in the Yaa position is post-translationally modified to hydroxyproline (Hyp) which enhances triple-helical stability. In humans, a family of at least 28 collagen types is present, each with type-specific biological and structural functions. The triple helical motif is also present in other proteins, such as macrophage scavenger receptors, collectins and C1q.
The most abundant collagens are the interstitial, fibril-forming collagens, particularly type I collagen. These collagens form the major tissue structures in animals through forming fibre bundle networks that are stabilized by specific cross-links to give stability and strength to the tissues. In contrast to the ‘major’ fibril forming collagens (types I, II and III) the ‘minor’ collagens are generally less broadly distributed and are typically found in particular tissue locations where the minor collagen may be a significant and critical component; e.g., type X collagen in hypertrophic cartilage or the type IV collagen in basement membranes.
Collagen has been shown to be safe and effective in a variety of medical products in various clinical applications (Ramshaw et al, J Materials Science, Materials in Science, (2009), 20(1) pg 3-8). For medical applications, collagen is generally used in two distinct formats. In one, intact tissue is used after chemical stabilisation, such as glutaraldehyde fixed porcine heart valves, for use in aortic valve replacement. The other is through preparation of purified soluble collagen which has been reconstituted into various products, such as dry, stabilised sheets or extruded fibres useful for wound dressings, adhesion barriers or devices for meniscal repair, with the processing giving the desired shape or form for the product. If necessary, the collagen device can be stabilised, either by chemical fixation, e.g., glutaraldehyde, or by a physical method e.g., dehydrothermal cross-linking. Purified soluble collagen has also been used extensively as a collagen paste for soft tissue augmentation and also for treatment of urinary incontinence. Reconstituted products are characterised by a high biochemical purity associated with low immunogenicity, controlled turnover, often over short time periods, controlled porosity and retention of cell-matrix interactions that are important in biological functions in tissues.
In order to purify collagen from animal collagenous tissues, typical methodologies include an initial digestion and solubilisation of the tissue through the use of an enzyme digestion step that removes the cross-linked regions while leaving the triple-helix intact. The solubilised collagen can then be purified to remove potential immunogenic contaminants. U.S. Pat. No. 6,548,077 for example describes a preparation of collagen from tissues involving contacting collagen with a first proteolytic enzyme followed by a reducing agent and a second proteolytic enzyme.
Addad et al (Mar. Drugs (2011), 9(6), 967-983) describe purifying collagen from jelly fish using acid-pepsin solubilisation of the tissue extract. However, in order to obtain a stable and useful final product a crosslinking step was required. Treatment with acid, and acetic acid in particular, leads to the swelling of tissues, and after pepsin digestion, gives a soluble collagen. The resultant soluble collagen product would be a weak non fibrous material that may need reconstituting into an insoluble format for many medical applications.
Alternative published methods include grinding the natural animal tissue that is rich in collagen to very fine particles that can be washed clean of impurities, either before or after processing into a useful medical material.
The majority of commercial quantities of collagen have been derived from animals such as bovine sources but with the concern of transmissible diseases, especially bovine spongiform encephalopathy (‘mad cow disease’) research effort has been spent on producing recombinant forms of collagen. Moreover, animal-derived collagen is limited in that extracted collagens cannot be designed and modified to enhance or change specific biological properties. Collagens are subject to extensive post-translational modifications both prior to and after deposition in the extracellular matrix. In particular, the fibrillar collagens are subjected to intra- and inter-molecular cross-linking that continues over the life of the molecule in the extracellular space. Thus, the amount of cross-linking present in collagens is influenced by, among other things, the age and physiology of the tissue from which the collagen is harvested. These differences influence both the extractability of collagens from tissue and the biophysical characteristics of these collagens. As a result, collagens isolated from tissues exhibit significant lot-to-lot variability and, as bulk materials, are often analytically intractable.
Accordingly, attention has shifted away from isolation of animal collagen and towards production of recombinant collagens.
Further, the use of recombinant DNA technology is desirable in that it allows for the potential production of synthetic collagens and collagen fragments which may include, for example, exogenous biologically active domains (i.e. to provide additional protein function) and other useful characteristics (e.g. improved biocompatability and stability).
Host systems such as yeast have been explored to recombinantly produce human coded collagen. However, yeast systems are complicated by the need to introduce genes for proline-4 hydroxylase to form the Hyp residues needed for stability of mammalian collagens. Typically, recombinant mammalian coded collagens are expressed in Pichia, which requires oxygen addition to get maximum hydroxylation, as well as methanol addition for induction, creating a need for enhanced, potentially flameproof engineering.
Other collagen-like material which does not require post translational modification has been sought as replacement to hydroxylated human collagen. Recently, research on bacterial genomes has indicated there are many putative bacterial proteins that contain Gly as every third residue and a high proline content, suggesting that collagen-like, triple-helical structures may be present in certain bacterial derived proteins (Peng Y et al (2010) Biomaterials 31(10):2755-2761; Yoshizumi A et al (2009) Protein Sci 18:1241-1251). Furthermore, several of these proteins have been shown to form triple-helices that are stable around 37° C., despite the absence of Hyp. The triple helical composition has been confirmed in a number of cases. Examples include cell surface proteins on certain bacterial cells and filaments on Bacillus anthracis spores. It has been postulated that expression of such collagen-like constructs in prophages present in pathogenic E coli strains appear to be responsible for dissemination of virulence-related genes through infection (Bella J et al (2012) 7(6) PLoS 1 e37872).
Use of recombinant technology, however, still has its shortcomings and hurdles. The use of host cells to produce the foreign proteins has added challenges such as the removal of contaminating host cell proteins whose presence in the final formulation of the desired proteins can result in adverse toxic or immunological reactions. If the recombinant protein is made intracellularly, the first step of a purification process involves lysis or disruption of the cell, which releases the contents of the cell into the homogenate and in addition produces subcellular fragments. If the recombinant protein is secreted out of the cell, the natural death of cells and release of intracellular host cell proteins into the supernatant can also give rise to toxic and immunogenic contamination. To remove these contaminants, many different purification steps are typically required. Affinity chromatography is commonly adopted to achieve high purity levels. This downstream processing is generally labour and resource intensive and cost prohibitive for large scale commercial production.
The large scale production of recombinant collagen-like proteins is still in its infancy. There are certain challenges that must be addressed with large scale production, including scalability of the process, production costs, complexity of the extraction method, compliance with GMP requirements, compliance with regulatory requirements, removal of contaminating host cell proteins, complexity of the purification method, suitability of the host cell. For example, human cell lines only result in moderate yields which are not suited to cost-effective, larger scale production.
Accordingly, there is a need for methods for the purification of recombinantly produced collagens, wherein such methods are cost-effective and which result in production of collagen in high yields and sufficient purity for various applications.